High fructose corn syrup (HCFS) is a widely used alternative to sucrose as a sweetener in many foods, and especially in soft drinks. High fructose corn syrup ultimately arises from the corn wet milling industry where starch is hydrolyzed, generally in a 2-stage process, to afford a starch hydrolyzate containing at least about 94% glucose, and the glucose is subsequently enzymatically isomerized to fructose using glucose isomerase (GI). Many variants of this process are known and have been successfully practiced for years.
The product of enzymatic isomerization of glucose is limited by equilibrium to about 50% fructose, whereas a high fructose corn syrup product approximately equal in sweetness to sucrose contains about 55% fructose. To attain the latter level commercially a portion of the product stream from enzymatic isomerization of glucose is sent to a separation unit, such as a chromatographic separation unit, to afford fractions containing about 90% fructose. These fructose-enriched fractions then are blended with the product stream to give the high fructose corn syrup of commerce.
Purified fructose, i.e., preparations containing at least 90% fructose, is a rather expensive commodity because the aforementioned separation process adds a substantial cost, especially when using a feedstock which is only about one-half fructose. Clearly there is an impetus for the production of fructose at levels greater than 50%, yet its equilibrium value from the isomerization of glucose cannot be significantly changed in a commercially practical matter. A 50% fructose content can be considered as a limitation inherent in the present method of HCFS production.
Several workers have reported that a microbial enzyme, mannose isomerase (MI), converts mannose to fructose with the equilibrium mixture containing about 70% fructose, which represents a substantial enrichment of fructose relative to the "glucose isomerase process" described above. Development of a "mannose isomerase process" alternative to fructose has been ignored, perhaps in part, because of the relative overabundance of corn and corn starch. But at least in principle a mannose isomerase process to fructose represents an option which circumvents the inherent limitations of a GI process.
However clear may be the principles involved, several problems need to be solved for an MI alternative to become commercially attractive. The microorganism producing mannose isomerase must grow rapidly in simple media and produce enzyme at a high level. The mannose isomerase must be able to be immobilized efficiently. The immobilized mannose isomerase should be quite specific as to substrate acted upon, should not need cofactors or be sensitive to inhibitors, and should have a reasonably long half-life under operating conditions. Finally, for a MI alternative process to be at all competitive it must be able to use a cheap, abundant feedstock which is geographically widely available.
Palleroni and Doudoroff [J. Biol. Chem., 218, 535 (1956)] appear to have first discovered a mannose isomerase from mutant strains of Pseudomonas saccharophila grown on fructose as a substrate, and reported that the equilibrium mixture contained 71% fructose. Although the enzyme did not require any cofactors, the MI did act on some aldose substrates other than mannose. Somewhat later, Takasaki [Agr. Biol. Chem., 31, 435 (1967)] reported that the mannose isomerase from Streptomyces aerocolorigenes isomerized mannose to afford 72-75% fructose at equilibrium, with the equilibrium constant being invariant with temperature over the range 1.degree.-40.degree. C. Hey-Ferguson and Elbein reported in J. Bacteriology, 101, 777 (1970) that the mannose isomerase from Mycobacterium smegmatis when grown on mannose isomerized the latter to afford 65% fructose at a pH optimum of 7.5. These investigators also reported the MI was active toward D-lyxnose. More recently, the patentees of U.S. Pat. No. 4,492,755 expanded on the prior work of Mayo et al. [ Carbohyd. Res., 8, 344 (1968)] by using mutants of Klebsiella aerogenes as well as species from the genera Escherichia and Lactobacillus which were constitutive MI producers to isomerize L-mannose to L-fructose. According to examples of the patentee the isomerization appears to require Co(II) ions during the isomerization.
Allenza reported that Pseudomonas cepacia produces a mannose isomerase intracellularly [P. Allenza, Ph.D. Dissertation, February, 1983, Univ. of Massachusetts, pp 69 to 75]. This microorganism was found to multiply rapidly on simple growth media under ordinary conditions to produce reasonably high levels of the enzyme, thus meeting two of the aforementioned criteria for an MI process. I have subsequently discovered that the mannose isomerase can be immobilized from unpurified whole cell extracts with high efficiency and without interference from other enzymes and without diminution of MI activity. The immobilized MI as prepared from unpurified whole cell extracts exhibits quite specific enzymatic activity and functions effectively either on solutions of purified mannose or, under appropriate circumstances, on a mannose containing feedstock widely available from the wood pulping industry and otherwise viewed as an industrial waste. Although mannose may constitute only about 40% of the dry solids of the latter feedstock, the high selectivity of the immobilized mannose isomerase as prepared from unpurified whole cell extracts described within effects the conversion of mannose to fructose without other detectable reactions. The immobilized MI needs no cofactors and does not appear to be inhibited by materials found or likely to be found in the feedstocks described above, and functions well in the absence of cobalt ions. Finally, the immobilized mannose isomerase as described within is sufficiently stable at operating conditions to be used over acceptable periods of time without change. In summary, I have appreciably enlarged the scope of knowledge pertinent to a mannose isomerase process alternative and have specified in detail means for economically producing fructose from cheap, abundant, and widely available feedstocks. My invention utilizes the inexpensive but highly efficient immobilization of crude mannose isomerase, an inexpensive source of mannose isomerase, and an inexpensive feedstock leading to the less expensive isolation of purified fructose. In its totality our invention provides the first economical mannose isomerase process alternative.